IntroductionIntroduction

Over the last decade, solid phase synthesis and combinatorial chemistry have undergone major evolution. In the early years, the process of split and mix revolutionized the concept of library synthesis, however, this strategy is less widely used, due in large measure to the fact that compound mixtures appeared to be problematic in terms of screening, while the alternative approach, single bead screening, suffers from many practical and synthesis problems not the least of these are the vast numbers of screens necessary when screening large split and mix libraries, and compound quantity. Thus to apply split and mix methodology effectively, efficient screening processes on single bead must be developed which allow the direct identification of the active bead and thus the active compound. Where release is required, it would be ideal if the cleavage could take place directly in a biologically compatible environment.

I. Linker synthesisI. Linker synthesis11

For this purpose, a specific “biologically” compatible safety-catch linker has been tailored to release amine based compounds for direct screening assays. The linker was synthesized in solution on a core based on proline and glutamic acid derivatives. Amine derivatives were pre-loaded on the linker before its attachment on Tentagel resin. The release mechanism based on the internal formation of a diketopiperazine was activated by reaction of the resin with TFA. Release was achieved in a phosphate buffer at pH 8 after activation of the safety-catch linker with TFA. Primary, secondary, aromatic amines and amidines led to high purity released material; only benzyl amines led to lower purity.

Utilization of a “Biological” Safety-Catch LinkerUtilization of a “Biological” Safety-Catch Linker for Direct Screening Assaysfor Direct Screening Assays

David Oraina and Mark Bradleyb,*

a) Current adress : Novartis Pharma AG, Lichtstrasse 35, WSJ-507.703, CH-4056 Basel, Switzerland,

E-mail : david.orain@pharma.novartis.com

b) University of Southampton, Department of chemistry, Highfield, Southampton SO17 1BJ, UK

www.soton.ac.uk/~bradley

II. Synthesis of TR inhibitorsII. Synthesis of TR inhibitors22

This new linker was used in the synthesis of known inhibitors of Trypanothione Reductase to demonstrate its potential to be used in a direct screening assay. This enzyme is key to the cell’s defence against oxidative stress, and is also present in the causative agents of the African sleeping sickness, leishmaniasis (skin lesions) and Chaga’s disease which is responsible of thousands of deaths each year on the north American continent.3 From a spermidine core loaded on the linker, three different inhibitors were synthesised by solid phase peptide synthesis. After activation of the safety-catch linker, cleavage in phosphate buffer afforded relatively pure material.

N

O

NH

O NH

O

O O N

OBoc R'

R

TG

HN

O NH TG

N

O

O

+

O

+NR'

R

HN

O

NH

O

O

O O N

OBoc R'

R

OH

N

O

NH

O O

OH

O N

O

NH

O O

O

O O O

O

NO2

Boc Boc

a)

NO

ON

O

OBoc

O O

CO2HH2N

+

Conditions : a) THF/H2O (1/1), Et3N 1.2eq, RT, 48h ; b) 4-HO-C6H4-CHO, DCC, DMAP, DCM, 18h, RT; c) NaBH4, Greenbromocresol, 20% HCl/EtOH; d) 4-NO2-C6H4-OCOCl, Py, DCM, 15 min, RT; e) R'RNH (1.1 eq), DIPEA (1 or 2 eq), DMF, 2-4h, RT; f) Pd(PPh3)4 (5% mol), pyrrolidine (1.5 eq), DCM, 10 min, RT; g) Tentagel-S-NH2, DIC, HOBt, DCM/DMF, O/N; h)TFA/DCM (1/1) 45 min; i) Phosphate Buffer, pH 8, 0.1M, 4 hours, 1 mL per 10 mg of resin.

b), c), d)

e), f)

g)h), i)

NH

O

O

O BocNON

O

BocHN-AA2-AA1-HN

BocHN-AA2-AA1-HN

OHN

NHH2N-AA2-AA1-HN

H2N-AA2-AA1-HN

NH

O

O

O BocNON

O

Fmoc-HN

Fmoc-HN

OHN

SPPS TG

TG

1) TFA/DCM (1/1), 45 min. RT

2) Phosphate Buffer 100mM, pH 8.00

a) AA1: Phe AA2: Pheb) AA1: Phg AA2: Trpc) AA1: Arg AA2: Trp

min6 7 8 9 10 11 12 13 14

mAU

0

250

500

750

1000

1250

1500

1750 Phe-Phe

min6 7 8 9 10 11 12 13 14

mAU

0

500

1000

1500

2000

2500Phg-Trp

min6 7 8 9 10 11 12 13 14

mAU

0

500

1000

1500

2000

Arg-Trp

Kinetics showed that the cleavage rate was dependent on the hydrophilicity of the lateral chain of the amino acids used. More interestingly,multiple releases could be achieved with this linker and so lead to the possibility of direct identification of the active compound without any other encoding techniques required.

Run

1 2 3 4

Release of inhibitors with run

0

0.2

0.4

0.6

0.8

1Phe-Phe

Phg-Trp

Arg-Trp

Re

lativ

e H

PL

C A

bso

rba

nce

Release of inhibitors with time

100 200 300

Re

lativ

e H

PL

C A

bso

rba

nce

0

0.2

0.4

0.6

0.8

1

Arg-Trp

Phg-Trp

Phe- Phe

Time (min)

III Direct enzymatic screeningIII Direct enzymatic screening

To determine the ability of this system to allow direct screening, a small library of 9 compounds was synthesized and cleaved individually. The crude solutions of inhibitors were tested directly on the enzymatic system. The inhibition rates were followed by consumption of NADPH at 340 nm or by generation of 2-nitrobenzothiol from the Ellman reagent at 415 nm. A good correlation wasobtained between the relative inhibition rates obtained in this study and previous works.4

0

25

50

75

100

TrpSerPhe

Arg

Asp

Lys

AA2

AA1

0

25

50

75

100

TrpSerPhe

Arg

Asp

Lys

AA2

AA1

% Inhibition recorded at 415 nm % Inhibition recorded at 340 nm

TSST NADPH

NADP+

NO2

SSNO2

SHNO2

2 TSH

Enzyme415 nm 340 nm

TGH2NTGN

HNH

O1) (BocNH-CH2CH2CH2)3CH-NCO,DIPEA, DCM

2) TFA/DCM

800 pmol/bead

1) Linker + spermidine scaffold2) SPPS

LinkerNBocHN-Phe-Arg(Pmc)-HN

BocHN-Phe-Arg(Pmc)-HN

TFA activation

Direct bead screening

TGNH

NH

O

HN

3

H2N

H2N

H2N

2 nmol/bead

Direct bead screening was then investigated on high-loading Tentagel beads. From Tentagel beads with a loading of 800 pmol/bead, a dendrimer was added to reach a level of loading around 2 nmol/bead. On these high-loading beads, the linker-spermidine scaffold was loaded and then the synthesis of the most active inhibitor from the previous screening was carried out. After activation of safety-catch linker with TFA, the beads were washed carefully before being spatially addressed in wells.

ConclusionConclusion

A new “biological” compatible safety-catch linker has been synthesized allowing a wide range of amines to be anchored. This linker has then been used in the synthesis of Trypanothione Reductase inhibitors. Direct screening assays with crude cleavage solution were then carried out and the inhibition rates were similar to previous works. On high-loading beads, direct one bead screening was realized and relatively good inhibition was achieved. Other dendrimers are being studied to reach higher bead capacity to allow a more efficient one bead screening library.

AcknowledgementsAcknowledgements

We thank the BBSRC (51/BO9512) for funding.

ReferencesReferences

11 Orain, D.; Bradley, M. Mol. Div. 2000, 5(1), 25.2 2 Orain, D.; Bradley, M. Tetrahedron Lett. 2001, 42, 515.3 3 Schirmer, R. H.; Muller, J. G.; Krauth-Siegel, R. L. Angew. Chem. Int. Ed. Engl. 1995, 34, 141.44 Smith, H. R.; Bradley, M. J. Comb. Chem. 1999, 1, 326.

0

20

40

60

80

100

123

340 nm

415 nm

Beads

% Inhibition for a direct bead screening

Different buffers were studied and best results were obtained when cleavage was carried out in a sodium tetraborate buffer 100 mM pH 8.0. Inhibition rates were measured at 340 nm and 415 nm, and the first results showed that 35% and 21% of inhibition were obtained on a single bead respectively.